The mechanisms by which eukaryotic organisms regulate the expression of their genetic information are important for basic scientific knowledge that is necessary for understanding many complex biological phenomena including human diseases. It is likely that eukaryotic organisms express their structural genes and regulate the level of expression by mechanisms qualitatively different from prokaryotic organisms. However, in eukaryotes, far less is understood about this process. The His3 gene of the yeast Saccharomyces cerevisiase is a simple eukaryotic gene that shows regulated expression as a function of the cell's physiological state. The proposed experiments employ genetic and molecular techniques to analyze this process in detail. The approach is similar in principle to that used successfully to elucidate prokaryotic regulatory mechanisms. A large number and wide variety of mutations within the His3 promoter/regulatory region will be isolated and characterized physically by DNA sequence analysis. Mutant DNAs will be introduced back into yeast such that each cell contains one copy of the mutated gene at the normal chromosomal position. Thus, the phenotypes conferred by these His3 derivatives will represent true in vivo phenotypes. For each mutation the level of His3 gene expression and the chromatin structure at the His3 locus will de determined under relevant physiological conditions. The experiments address five main points: 1) The relationship between chromatin structure and transcription. 2) The nature of a eukaryotic promoter. 3) The nature of a eukaryotic regulatory site. 4) The interactions of regulatory proteins and regulatory sites. 5) The mechanism of general amino acid biosynthetic control.